Several publications and patent documents are referenced in this application by author name and year of publication in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these reference can be found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
The plastid genetic system of higher plants is highly polyploid. For example, in a tobacco leaf there are as many as 100 chloroplasts, each carrying ˜100 identical genome copies, a total of 10,000 copies in a leaf cell. High-level protein expression, lack of pollen transmission and the feasibility to engineer polycistronic expression units make the plastid genome an attractive alternative to nuclear engineering. Plastid transformation vectors often contain a selective marker, most commonly a spectinomycin resistance (aadA) gene, flanked by plastid DNA sequences targeting insertion of the marker gene by homologous recombination into the plastid gnome. Genes of commercial value but lacking a selectable phenotype are physically linked to the selective marker and the two genes are integrated together as a block of heterologous sequences. Plastid transformation is accomplished by biolistic DNA delivery or polyethylene glycol induced uptake of the transforming DNA followed by selection for the antibiotic resistance marker to ensure preferential propagation of plastids with transformed genome copies. As the result, all the 10,000 wild-type plastid genome copies in a cell are replaced with transgenic copies during a gradual process (Maliga, 1993).
Incorporation of a selectable marker gene is essential to ensure preferential maintenance of the transformed plastid genome copies. However, once transformation is accomplished, maintenance of the marker gene is undesirable. One problem may be the metabolic burden imposed by the expression of the selectable marker gene. For example FLARE-S, the product of the marker gene with good prospects to transform cereal chloroplasts, accumulates up to 18% of the total soluble cellular protein (Khan and Maliga 1999). The second problem is the relatively high potential for horizontal transfer of plastid marker genes to microbes (Tepfer 1989; Dröge et al. 1998; Sylvanen 1999), as commonly used plastid maker gene constructs are efficiently expressed in E. coli (Carrer et al. 1993; Svab and Maliga 1993). Therefore, having plastid marker genes in commercial products is undesirable.